Revision as of 16:15, 15 September 2015

P_phoA -I13504- phosphate responsive promoter with GFP generator

Biology of P_phoA

Fig.1 Phosphate sensing mechanism of P_phoA.

Escherichia coli (E. coli) detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, P_phoA is cross-regulated by PhoB and PhoR. The sensory histidine kinase PhoR behaves either as an activator or inactivator for PhoB depending on different states (inhibition state, activation state, deactivation state). When phosphate is limited, PhoR acts as a phospho-donor for the autophosphorylation of PhoB. The phosphorylated PhoB will directly activate P_phoA. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates P_phoA.

Constructs for characterization

Fig.2 Phosphate sensing construct with reporter.

With the phosphate (pho) regulon from E. coli, it can be utilized for detecting phosphate level. To make a phospahte-sensing device, we obtained the promoter, P_phoA, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different phosphate level can be detected and characterized.

RFU measurement

Fig.3 Activity of P_phoA in E. coli DH10B in different phosphate concentrations

As shown in Figure 3, P_phoA is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of P_phoA is from 0-200 μM of phosphate.

Sequence and Features